Mycobacterium tuberculosis complex lineage 5 exhibits high levels of within-lineage genomic diversity and differing gene content compared to the type strain H37Rv.

Pathogens of the Mycobacterium tuberculosis complex (MTBC) are considered to be monomorphic, with little gene content variation between strains. Nevertheless, several genotypic and phenotypic factors separate strains of the different MTBC lineages (L), especially L5 and L6 (traditionally termed Mycobacterium africanum) strains, from each other. However, this genome variability and gene content, especially of L5 strains, has not been fully explored and may be important for pathobiology and current approaches for genomic analysis of MTBC strains, including transmission studies. By comparing the genomes of 355 L5 clinical strains (including 3 complete genomes and 352 Illumina whole-genome sequenced isolates) to each other and to H37Rv, we identified multiple genes that were differentially present or absent between H37Rv and L5 strains. Additionally, considerable gene content variability was found across L5 strains, including a split in the L5.3 sub-lineage into L5.3.1 and L5.3.2. These gene content differences had a small knock-on effect on transmission cluster estimation, with clustering rates influenced by the selected reference genome, and with potential overestimation of recent transmission when using H37Rv as the reference genome. We conclude that full capture of the gene diversity, especially high-resolution outbreak analysis, requires a variation of the single H37Rv-centric reference genome mapping approach currently used in most whole-genome sequencing data analysis pipelines. Moreover, the high within-lineage gene content variability suggests that the pan-genome of M. tuberculosis is at least several kilobases larger than previously thought, implying that a concatenated or reference-free genome assembly (de novo) approach may be needed for particular questions.

Phylogenomics of Mycobacterium africanum reveals a new lineage and a complex evolutionary history.

Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC). The MTBC comprises several human-adapted lineages known as M. tuberculosis sensu stricto, as well as two lineages (L5 and L6) traditionally referred to as Mycobacterium africanum. Strains of L5 and L6 are largely limited to West Africa for reasons unknown, and little is known of their genomic diversity, phylogeography and evolution. Here, we analysed the genomes of 350 L5 and 320 L6 strains, isolated from patients from 21 African countries, plus 5 related genomes that had not been classified into any of the known MTBC lineages. Our population genomic and phylogeographical analyses showed that the unclassified genomes belonged to a new group that we propose to name MTBC lineage 9 (L9). While the most likely ancestral distribution of L9 was predicted to be East Africa, the most likely ancestral distribution for both L5 and L6 was the Eastern part of West Africa. Moreover, we found important differences between L5 and L6 strains with respect to their phylogeographical substructure and genetic diversity. Finally, we could not confirm the previous association of drug-resistance markers with lineage and sublineages. Instead, our results indicate that the association of drug resistance with lineage is most likely driven by sample bias or geography. In conclusion, our study sheds new light onto the genomic diversity and evolutionary history of M. africanum, and highlights the need to consider the particularities of each MTBC lineage for understanding the ecology and epidemiology of TB in Africa and globally.

Storage of Sputum in Cetylpyridinium Chloride, OMNIgene.SPUTUM, and Ethanol Is Compatible with Molecular Tuberculosis Diagnostic Testing.

We compared cetylpyridinium chloride (CPC), ethanol (ETOH), and OMNIgene.SPUTUM (OMNI) for 28-day storage of sputum at ambient temperature before molecular tuberculosis diagnostics. Three sputum samples were collected from each of 133 smear-positive tuberculosis (TB) patients (399 sputum samples). Each patient's sputum was stored with either CPC, ETOH, or OMNI for 28 days at ambient temperature, with subsequent rpoB amplification targeting a short fragment (81 bp, GeneXpert MTB/RIF [Xpert]) or a long fragment (1,764 bp, in-house nested PCR). For 36 patients, Xpert was also performed at baseline on all 108 fresh sputum samples. After the 28-day storage (D28), Xpert positivity did not significantly differ between storage methods. In contrast, higher positivity for rpoB nested PCR was obtained with OMNI (n = 125, 94%) than with ETOH (n = 114, 85.7%; P = 0.001). Smears with scanty acid-fast bacilli (AFB) had lower rpoB PCR positivity with ETOH storage (n = 10, 41.7%) than with CPC (n = 16, 66.7%; difference, 25%; 95% confidence interval [CI], 3.5 to 46.5; P = 0.031) or OMNI (n = 16, 69.6%; difference, 26.1%; 95% CI, 3.8 to 48.4; P = 0.031), with no difference between CPC and OMNI. Poststorage, the threshold cycle (CT ) values significantly decreased compared to those prestorage with ETOH (difference, -1.1; 95% CI, -1.6 to -0.6; P = 0.0001) but not with CPC (P = 0.915) or OMNI (P = 0.33). For one patient's ETOH- and CPC-stored specimens with a CT of <10, Xpert gave results of rifampin false resistant at D28, which was resolved by repeating Xpert on a 1/100 diluted specimen. In conclusion, 28-day storage of sputum in OMNI, CPC, or ETOH at ambient temperature does not impact short-fragment PCR (Xpert), including for low smear grades. However, for long-fragment PCR, ETOH yielded a lower PCR positivity for low smear grades, while the performance of OMNI and CPC was excellent for all smear grades. (The study has been registered at ClinicalTrials.gov under registration number NCT02744469.).

Low sensitivity of the MPT64 identification test to detect lineage 5 of the Mycobacterium tuberculosis complex.

PURPOSE: Differentiation of the Mycobacterium tuberculosis complex (MTBc) from non-tuberculous mycobacteria (NTM) is important for tuberculosis diagnosis and is a prerequisite for reliable phenotypic drug-resistance testing. We evaluated the performance of the rapid MPT64 antigen identification test for the detection of Mycobacterium africanum lineage 5 (MAF L5). METHODOLOGY: Smear-positive tuberculosis patients' sputa were included prospectively. Culture was performed on Löwenstein-Jensen medium and, when positive, the MPT64 test and the classical para-nitro benzoic acid susceptibility and heat-labile catalase (PNB/catalase) identification tests were performed. The MPT64 test was repeated 14 days after an initially negative first testing. Direct spoligotyping was performed for MTBc lineage determination. RESULTS: In total, 333 isolates were tested for all of the methods. Three hundred and twenty-two (96.7 %) were pure MTBc, by agreement between spoligotyping and PNB/catalase, and 11 were NTM or a mixture of MTBc/NTM. The MPT64 test conducted on day zero of culture-positivity correctly identified most of the pure MTBc isolates (93.2 %, 300/322), but it failed to detect 24 % of the L5 isolates (18/75) versus 2 % (4/202) of the L4 ones [OR=15.6 (5.3-45.8), P<0.0001], with improved sensitivity for L5 detection on repeat testing after 14 days. The L5-wide non-synonymous single-nucleotide polymorphism in the mpt64 gene may explain the poor performance of the MPT64 test for L5. CONCLUSION: The MPT64 test has a lower sensitivity for detecting L5 isolates of the MTBc, and can be considered as a first-screening test that should be confirmed by another identification method when it produces negative results in countries with L5. Given the microbiological bias in both the isolation and identification of MAF lineages, diagnostics with high sensitivity for direct testing on clinical material are preferable.

Genotypic characterization directly applied to sputum improves the detection of Mycobacterium africanum West African 1, under-represented in positive cultures.

BACKGROUND: This study aimed to compare the prevalence of Mycobacterium tuberculosis complex (MTBc) lineages between direct genotyping (on sputum) and indirect genotyping (on culture), to characterize potential culture bias against difficult growers. METHODOLOGY/PRINCIPAL FINDINGS: Smear-positive sputa from consecutive new tuberculosis patients diagnosed in Cotonou, (Benin) were included, before patients had started treatment. An aliquot of decontaminated sputum was used for direct spoligotyping, and another aliquot was cultured on Löwenstein Jensen (LJ) medium (90 days), for indirect spoligotyping. After DNA extraction, spoligotyping was done according to the standard method for all specimens, and patterns obtained from sputa were compared versus those from the derived culture isolates. From 199 patient's sputa, 146 (73.4%) yielded a positive culture. In total, direct spoligotyping yielded a pattern in 98.5% (196/199) of the specimens, versus 73.4% (146/199) for indirect spoligotyping on cultures. There was good agreement between sputum- and isolate derived patterns: 94.4% (135/143) at spoligotype level and 96.5% (138/143) at (sub)lineage level. Two of the 8 pairs with discrepant pattern were suggestive of mixed infection in sputum. Ancestral lineages (Lineage 1, and M. africanum Lineages 5 and 6) were less likely to grow in culture (OR = 0.30, 95%CI (0.14 to 0.64), p = 0.0016); especially Lineage 5 (OR = 0.37 95%CI (0.17 to 0.79), p = 0.010). Among modern lineages, Lineage 4 was over-represented in positive-culture specimens (OR = 3.01, 95%CI (1.4 to 6.51), p = 0.005). CONCLUSIONS/ SIGNIFICANCE: Ancestral lineages, especially M. africanum West African 1 (Lineage 5), are less likely to grow in culture relative to modern lineages, especially M. tuberculosis Euro-American (Lineage 4). Direct spoligotyping on smear positive sputum is effective and efficient compared to indirect spoligotyping of cultures. It allows for a more accurate unbiased determination of the population structure of the M. tuberculosis complex. TRIAL REGISTRATION: ClinicalTrials.gov NCT02744469.